Prediction of improved antimalarial chemotherapy of artesunate-mefloquine in combination with mefloquine sensitive and resistant Plasmodium falciparum malaria

Background Declining in susceptibility of Plasmodium falciparum to mefloquine is reported in South-East Asia. A revisiting on mefloquine pharmacokinetics-pharmacodynamics (PK/PD) could assist in finding new appropriate dosage regimens in combination with artesunate as a three-day course treatment. Objective This study aimed to investigate promising alternative artesunate-mefloquine combination regimens that are effective for the treatment of patients with mefloquine-sensitive and resistant P. falciparum malaria. Methods Data collected during 2008–2009 from 124 patients with uncomplicated P. falciparum malaria were included in the analysis, 90 and 34 patients with sensitive and recrudescence response, respectively. All patients were treated with a three-day combination of artesunate-mefloquine. Population PK-PD models were developed. The developed models were validated with clinically observed data. Simulations of clinical efficacy of alternative mefloquine regimens were performed based on mefloquine sensitivity, patients’ adherence and parasite biomass. Results The developed PK/PD models well described with clinically observed data. For mefloquine-resistant P. falciparum, a three-day standard regimen of artesunate-mefloquine is suitable (>50% efficacy) only when the level of parasite sensitivity was < 1.5-fold of the cut-off level (IC50 < 36 nM). For mefloquine-sensitive parasite with IC50 < 23.19 nM (0.96-fold), all regimens provided satisfactory efficacy. In the isolates with IC50 of 24 nM, regimen-I is recommended. Curative treatment criteria for mefloquine and artesunate were C336h (>408 ng.mL-1) or Cmax/IC50 (>130.1 g.m/M), and Cmax/IC50 (>381.2 g.m/M), respectively. Conclusions Clinical use of a three-day standard artesunate-mefloquine is suitable only when the IC50 of P. falciparum isolates is lower than 36 nM. Otherwise, other ACT regimens should be replaced. For mefloquine-sensitive parasite, a dose reduction is recommended with the IC50 is lower than 23.19 nM.


Introduction
Malaria-related mortality has tremendously reduced during the last two decades since the introduction of artemisinin-based combination therapy (ACT) [1]. Artesunate-mefloquine is one of the commonly used ACT for the first-line treatment of uncomplicated Plasmodium falciparum in Southeast Asia and Africa [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. In South-East Asia, its clinical efficacy has been continuously declined, with failure rates of 10-30% [1]. The highest failure rate was reported in the Thai-Myanmar border areas (42-day cure rate of 72.58%) in 2010 [18]. This high failure rate was attributed to pharmacokinetic factors (inadequate drug concentrations) and reduced susceptibility or resistance of P. falciparum to either mefloquine or artesunate, or both. Inadequate blood concentrations of mefloquine was shown to significantly influence treatment response compared with artesunate. Notably, the current three-day course regimen may not provide adequate drug concentrations in the resistant parasites. Revisiting the pharmacokinetic and pharmacodynamic relationship of drug concentration-time profile and clinico-parasitological response following this combination therapy may offer effective alternative dosage regimens for both mefloquine-sensitive and resistant P. falciparum. The pharmacokineticpharmacodynamics (PK/PD) modelling has been successfully applied to predict the appropriate dosage regimens of various antimalarial drugs for a clinical use [19][20][21][22][23], exemplified by the SJ733 (an oral ATP4 inhibitor) [23]. The current study aimed to investigate promising alternative regimens of artesunate-mefloquine with improved efficacy to cope with multidrug resistant P. falciparum using PK/PD model approach.

Materials and methods
The flow-chart of study framework is shown in Fig 1.

Data source and study population
Data from the previously published article on the clinical efficacy of the three-day artesunatemefloquine combination in the Thai-Myanmar borders during 2008-2009 were used for pharmacokinetic-pharmacodynamic (PK/PD) analysis [18]. All patients were diagnosed with uncomplicated P. falciparum malaria. In brief, 124 patients (aged 16-50 years) were included in the study, 90 and 34 patients with sensitive and recrudescence response, respectively. All received 200 mg of artesunate and 750 mg of mefloquine on day 1, followed by 200 mg of artesunate, and 500 mg of mefloquine on day 2, followed by 30 mg of primaquine on day 3.

Population PK/PD modeling
The PK/PD models for mefloquine and artesunate/dihydroartemisinin (active metabolite of artesunate) were constructed using nonlinear mixed-effects modeling (MonolixSuite software, version 2021R1, Antony, France; Lixoft SAS, 2021). Pharmacokinetic parameters were estimated using the build-in stochastic approximation expectation-maximization algorithm. Various compartment model with different order absorption and elimination were performed to fit with drug concentration-time data. Pharmacokinetic parameters were normally distributed when transformed to log-scale. The pharmacodynamic model was evaluated using E max model (turn-over rate) with production inhibition. The model was corrected with the fraction of unbound drug (f u ) in plasma and tissue and was tested for sigmoidicity characteristics. Pharmacodynamic parameters were tested following non-transformed and log-normal transformation. The pharmacodynamic equation is shown below; Where parasite is number of parasite; C is drug plasma-concentration profiles; IC 50 is the half maximal inhibitory concentration that inhibit parasite growth by 50%; K in is indirect turnover model with full inhibition of production; K out is the degradation rate of parasite. The residual variability and the types of error models were evaluated using proportional, constant, and combined error models with power law. Predefined criteria for a model selection were: (i) the decrease in minimum objective function value (OFV), Akaike Information Criteria (AIC), Bayesian Information Criteria (BIC), and Corrected Bayesian Information Criteria (BICc), and (ii) the percentage of root mean square errors (RSE%), Graphical Goodness of Fit (GOF) including the observed versus predicted concentrations, scatter plot of residual, and the virtual predictive check (VPC). A significant level for the inclusion of the covariates (age, sex, bodyweight, and mefloquine level before treatment) in the model was set at α = 0.05. The plotting of model-based individual prediction (IPRED) and population prediction (PRED) versus observed concentrations (GOF) was used for model evaluation. VPC included the observational data versus simulated data (1,000 patients) with the 10th, 50th, and 90th percentiles.

PK/PD model validation
The IC 50 (concentration that inhibits parasite growth by 50%) reflecting parasite's susceptibility to mefloquine from the clinical study in the same areas areas [24] were used for model validation (IC 50 12.9, 19.1 and 30.4 nM for isolates from Ranong, Kanchanaburi and Ratchaburi, and Tak provinces, respectively). These IC 50 values were used to report the drug's efficacy in those area (Ranong, Kanchanaburi and Ratchaburi, and Tak province). The evaluation of drug's efficacy were described in the next section. The pharmacodynamic endpoint were the 42 days curative rate.

Monte Carlo simulations
The final PK/PD (1,000 virtual patients with 10 simulations) models were used to simulate optimal dosages of mefloquine in the combination regimens that provided high clinical efficacy using Monte Carlo simulations (Simulix version 2021R1, Antony, France; Lixoft SAS, 2021). The simulated dose regimens (oral administration) for mefloquine-resistant P. falciparum included: (i) 750 mg on day 1 (day 0), followed by 500 mg on day 2, (ii) 500 mg once daily for 42 days, (iii) 500 mg every 72 hours for 42 days, (iv) 500 mg every 96 hours for 42 days, and (v) 250 mg every 12 hours for 42 days. The simulated regimens for mefloquine-sensitive strains were: (i) 750 mg on day 1 and 500 mg on day 2, (ii) 500 mg on day 1 and 250 mg on day 2, (iii) 750 mg on day 1, (iv) 500 mg on day 1, and (v) 250 mg on day 1, 2, and 3. The simulated regimens are based on trials and erros until it provides the curative rate close to 100%.

Effect of patients' adherence
Due to long course treatment of the proposed reimen (42 days), the effect of patients' adherence to medication (i.e., 100%, 70%, 50%, and 30%) on treatment efficacy were evaluated.

Toxicity approach
Due to the permanent neurological deficits following standard treatment, the relative risk of plasma/blood concentration over the threshond, resulting in the disruption of calcium homeostasis and ER function following five regimens, were calculated.

Establishment of the criteria for curative treatment
Receiving Operating Characteristic (ROC) curves were used to assess the accuracy of the following cut-off parameters for curative outcome: area under the curve during the first 7 days (AUC 0-7days ), trough plasma/whole-blood concentration at 336 h (C trough,336h ), and peak concentration and IC 50 ratio (C max /IC 50 ). It is noted that the mechanism of action of a mefloquine is on the blood stage (blood schizontocide), curative parameter "C trough, 336h (14 days)" covers the blood stage. A binomial proportion (Wilson/Brown) method was performed at a statistically significant level of 0.05 (GraphPad Prism version 9.20 for Windows, GraphPad Software, La Jolla California USA). Sensitivity, specificity, accuracy, negative predictive value (NPV), positive predictive value (PPV), positive likelihood ratios (LR+), negative likelihood ratios (LR-), and diagnostic odd ratios were calculated for an internal validity.

PK/PD models
A one-compartmental model with zero-order kinetic absorption and linear elimination showed the best characterized (best fit) the population pharmacokinetic properties for mefloquine. A transit-compartment model with one compartment and linear elimination best characterized (best fit) the pharmacokinetic properties of artesunate and dihydroartemisinin. The pharmacokinetic model of artesunate/dihydroartemisinin was in accordance with that previously reported [a transit-compartment model followed by one-compartment disposition] [26]. Since both artesunate and dihydroartemisinin are almost immediately eliminated from the systemic circulation, their contribution to parasite elimination is only during the first two days of treatment. Therefore, the neither artesunate nor artesunate could be fit with parasite clearance throughout 42 days. With a short-half-life, primaquine unlikely plays important role for parasite elimination due to fast elimination, particulary of blood stage. Only mefloquine was therefore used for PK/PD modeling and simulation as it play role in parasite elimination for the whole 42-day follow up. The final PK/PD model of mefloquine was well characterized by E max model (turn-over rate) without sigmoidicity when corrected with f u . The inclusion of body weight and sex did not improve model quality. All parameters in all models showed low to moderate variation (%RSE). The analysis of OFV, AIC, BIC, BICc and GOF were summarized in the S1 File. Final population parameters estimated for mefloquine (resistance and sensitive), artesunate and dihydroartemisinin are shown in Tables 1 and 2, respectively.

Model validation
The developed PK/PD models using the IC 50 of 12.9, 19.1 and 30.4 nM adequately predicted the reported clinical efficacy of mefloquine [19]. The efficacy predicted based on the IC 50 of 23.19 (sensitive) and 63.84 nM (resistance) reported for the current study was 74 and 13.8%, respectively [18].

PK/PD simulations for prediction of the clinical efficacy of mefloquine
Mefloquine-resistant P. falciparum. Without the effect of patients' adherence (100%), the efficacy of mefloquine for regimen-I (standard regimen) ranged from 1 to 57.3% (Fig 2), with IC 50 ranging from 1 to 5-fold (24-110 nM). All other regimens provided better treatment efficacy than regimen-I (p � 0.001). Regimen-V was the most effective (70.6-98.4% cure) (Fig  2). Number-needed-to-treat (NNT) and relative risk (RR) for regimen-V were 1.44-2.43 and 0.04-0.58, respectively. With a 1 to 2-fold increase in IC 50 (24-72 nM), all proposed regimens provided moderate efficacy (>50%). With a 3 to 5-fold increase in IC 50 , the efficacy of regimen-III and IV dramatically dropped to around 30 and 15%, respectively. The efficacy of regimen-II and V were high (>70%). The simulated C max ratios between regimen-II, III, IV and V compared with regimen-I were 5.65, 1.82, 1.44, and 5.55-fold, respectively. Since regimen-II and V provided C max over 5-fold of the standard regimen, patients may be at risk of toxicities. Regimen-II and V were inappropriate for clinical use, and therefore, the effect of patients' adherence was evaluated only for regimen-III, and IV. Treatment efficacy, RR, and NNT for all regimens when the adherence was 100% are presented in Figs 2-4 (2, 3, and 4 for efficacy, RR, and NNT, respectively). With 70% adherence, regimen-III and IV provided improved efficacy compared with regimen-I for isolates with different levels of sensitivity to mefloquine (p<0.001) for mefloquine resistance (S1 File).The NNT and RR were 3.21-43.68 and 0.12-0.81, respectively. When the IC 50 was 1 to 1.5-fold of the cut-off value, the efficacy of regimen-III and IV were moderate (50-70%) and high (>70%), respectively. When the IC 50 was increased by 2-fold, regimen-IV provided low efficacy (43.9%) efficacy. When the IC 50 was increased by 5-fold, the efficacy for regimen-III and IV were 13.3 and 7.2%, respectively (S1 File).
With 50% adherence, both regimen-III and IV still provided superior efficacy than regimen-I (α = 0.001). Only a 1-fold increase of IC 50 resulted in moderate efficacy (>50%) for both regimen-III and IV. When the IC 50 was increased by 1.5-fold, treatment efficacy of regimen-III was still over 50% (NNT = 5.05, p<0.001). With a 2 to 3-fold increase in IC 50 , the efficacy was 15-50% for both regimens. With a 5-fold increase in IC 50 , the efficacy of both regimens were lower than 10%.
With 30% adherence, the efficacy of regimen-III appeared to be slightly higher than regimen-I (S1 File). However, no significant difference was found for 1-fold (p = 0.2), 1.5-fold (p = 0.09), and 2-fold (p = 0.02) increase in IC 50 . The efficacy of regimen-IV for all sensitivity  levels except 5-fold was lower than regimen-I. No significant difference was found between regimen-I and IV when the IC 50 was increased by 3-fold (p = 0.08) and 5-fold (p = 0.24). The efficacy and RR of each regimen at 100 and 30% adherence are shown in Figs 5 and 6. The predicted mefloquine plasma/blood concentration profiles for each regimen are shown in S1 File.

Predicted mefloquine in malaria
Five different levels of parasite biomass [i.e., 30,000 (group-I), 20,000 (group-II), 15,000 (group-III), 10,000 (group-IV), and 5,000 (group-V)] with three different levels of parasite sensitivity to mefloquine were simulated. Overall, treatment efficacy seemed to be an inverse relationship with parasites biomass. All scenarios in all groups except for the IC 50 of 63.83 nM in group-II (p = 0.1) showed a significant difference in efficacy compared with group-I (p<0.001) (Fig 7), with odds ratios of 0.02-0.58 (Fig 8). For all parasite sensitivity levels, the group-V with IC 50 of 30.89 nM was the most effective regimen (75.30% cure). In contrast, the group-I with IC 50 of 63.84 nM was the least effective (1.3% cure).

Mefloquine sensitive P. falciparum
Three levels of mefloquine sensitivity based on clinical IC 50 and two varied IC 50 values were simulated assuming 100% patients' adherence. Overall, the efficacy of all regimens was inferior to regimen-I for all scenarios (p = 0.001). However, the efficacy of all regimens except regimen-IV with IC 50 of 23.19 nM (49.7%) were still higher than 50% (moderate efficacy). With  10% (77.1, 65.0, 61.6, 53.7 and 64.6%). Notably, treatment efficacy was dramatically decreased to around 60% when the IC 50 was increased to 23.19 nM (73.9, 60.2, 56.7, 49.7 and 60.6%). Comparison of clinical efficacy of regimen-I and other regimens for all IC 50 are provided in S1 File. Comparison of treatment efficacy between the 0.25-fold and 0.96-fold IC 50 with different regimens are shown in Fig 9. The predicted mefloquine plasma/blood concentration profiles for each regimen are shown in S1 File.

Discussion
The emergence and spread of mefloquine resistance have led to a decrease in clinical efficacy of artesunate-mefloquine combination. For mefloquine-resistant P. falciparum, a three-day artesunate-mefloquine regimen (regimen-I) should be replaced by other regimens when the IC 50 of mefloquine was higher than 36 nM (1.5-fold of the cut-off level) (<50% efficacy). It was clear that regimen-II/V provided the best efficacy for P. falciparum with different sensitivity. These regimens were the best option for mefloquine-resistant P. falciparum. Nonetheless, these proposed regimens resulted in a 5-fold increase in C max compared with regimen-I. Clinical use of regimen-II or V may result in an increased risk of mefloquine toxicity. In such case, regimen-III or IV would be a preferable choice for mefloquine-resistant parasite with IC 50 lower than 48 nM, particularly when supervised medication or directly observed therapy (DOT) is applicable. Notably, it is clear that patients' adherence significantly affected the treatment efficacy of the long-course treatment regimens (42 days). Treatment efficacy for parasite strains with IC 50 below 48 nM dramatically dropped to lower than 50% when the adherence was only 30%. Interestingly, initial parasite biomass significantly affected the clinical efficacy of mefloquine in the standard regimen (I), the higher initial parasite biomass, the higher treatment failure rate. The current standard regimen is thus, only suitable when the initial parasite biomass was lower than 5,000/μL (IC 50 : 30.89-32.71 nM). For mefloquine-sensitive parasite,  although the clinical efficacy of all proposed regimens was relatively low compared with regimen-I (moderate efficacy). Ideally, regimen-IV with the lowest mefloquine total dose (500 mg) would be a preferable choice considering the amount of dosage administration. This regimen, however, provided the lowest efficacy compared with others. Its efficacy was also decreased to 50% for the parasite strains with IC 50 of 23.19 nM (Fig 5). Regimen-II, III and V had a comparable amount of mefloquine dose as well as clinical efficacy. In cases when patients' adherence to medication is of great concern, a single dose regimen-III (on day 1) is a preferable choice. Due to Mefloquineinduced neurotoxicity, low dose regimens, i.e., regimen-II and V are preferable choices. Remarkably, the efficacy of all proposed regimens for the parasite strains with IC 50 of 23.19 nM was decreased to 60%. In such case, Regimen-I is more appropriate. Similarly to the resistant strains, initial parasite biomass significantly influenced the efficacy of mefloquine in the sensitive isolates. When the IC 50 of mefloquine was between 13.10 and 19.39 nM, regimen-I was still effective with an initial parasite biomass of lower than 10,000/μL. In cases when the initial parasite density was over 10,000/μL, clinical use of this regimen may not be effective (<50% efficacy).
Besides the problem with mefloquine-resistant P. falciparum in the Great Mekong Subregion (GMS), the emergence and spread of resistance to artemisinin-based therapy is the great concern in all malaria-endemic areas. The combination of two partner drugs with different mechanisms of action (triple artemisinin-based combination therapy or TACT) is an optional choice for malaria treatment [27,28]. It is noted that this approach has been successfully applied in HIV as well as tuberculosis [27]. The first clinical trial of TACT showed that TACT could tackle artemisinin-based resistance problems with high efficacy, safety and tolerability [28]. TACT is, therefore, a promising approach for malaria therapy.
Few clinical information have been corrected based on previous study [18], therefore, the effects of covariate parameters on models are limited. Since the highest mefloquine resistance level is reported in Thailand [1], the proposed curative criteria based on clinical data in Thailand could be applied for the treatment of uncomplicated P. falciparum malaria in other endemic areas. The efficacy reported in this study [18] was 78.5%, while the efficacy in other Southeast Asia countries (Cambodia, Myanmar, and Laos) [4][5][6][7][8][9][10][11][12], and African countries (Mali, and Senegal) [13][14][15][16][17] ranged 96 to 100%. However, clinical application based on the results of the current study may be limited as the PK/PD models did not include the effect of artesunate and dihydroartemisinin on parasite clearance. Furthermore, the impact of initial parasite biomass on the clinical efficacy of regimen-I artesunate-mefloquine combination seemed to be underestimated (low efficacy). An increase in initial parasite biomass has been reported to result in delayed parasite clearance [29]. Therefore, simulation of efficacy for the 42-day follow up may not be long enough to capture such effect. Moreover, external validation for curative treatment criteria using separate data sets was not performed. Further large clinical trials of the three-day artesunate-mefloquine combination with different levels of initial parasite biomass is required. Also, only nausea and vomiting have been reported in patients and no other neurological deficits have been reported [18]. The accumulation of this drug exceeding 50 μM following standard treatment in some patients, however, would disrupt the function of neuronal calcium homeostasis and ER functions [30]. Results based on this simulation showed that there were not significantly different of predicted brain-concentration profiles between patients with and without adverse events following a standard treatment (p = 0.2734). Nevertheless, the RR of mefloquine concentrations over threshold of the disruption of neuronal calcium homeostasis for each regimen compared with standard treatment were reported for a risk-benefit assessment (All regimens are at risk of the disruption of neuronal calcium homeostasis).

Conclusions
In conclusion, clinical use of a three-day artesunate-mefloquine (regimen-I) is suitable only when the IC 50 of P. falciparum isolates is lower than 36 nM. With the resistant strains (IC 50 up to 48 nM), two proposed regimens (III and IV) are preferable under DOT therapy or supervised medication. Otherwise, other ACT regimens should be replaced as the clinical efficacy would be dramatically decreased if patients' adherence to medication is poor. For mefloquinesensitive parasite, all regimens should provide satisfactory efficacy if the IC 50 is lower than 23.19 nM. With decreased parasite sensitivity (IC 50 close to 24 nM), a three-day artesunatemefloquine is recommended since the efficacy of all proposed regimens would be extremely reduced to lower than 60%.